Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide

Abstract

Objective To explore the effect of zinc finger protein A20 on senescence and apoptosis in human degenerative nucleus pulposus cells. Methods Human degenerative nucleus pulposus cells were extracted and cultured. Small interference RNA (siRNA) decreased the expression of A20. Lipopolysaccharide (LPS) was used as an inflammatory inducing factor. Control group, LPS group, siRNA-A20 group and siRNA-A20+LPS group were set up. The protein expression levels of A20, P21, P16, BAX, BID, BCL2, P65 and P-P65 were detected by Western blotting. β-galactosidase was detected by aging β-galactosidase staining kit, and Annexin V-PI double labeling and TUNEL staining was employed to detect apoptosis and to explore the effect of zinc finger protein A20 on senescence and apoptosis of nucleus pulposus cells. Caffeic acid phenethyl ester (CAPE) was used in rescue experiment, and the protein expression levels of A20, P21, P16, BAX, BID, BCL2, P65 and P-P65 were detected to explore the possible pathway of cell senescence and apoptosis induced by down-regulation of zinc finger protein A20. Results Compared with the control group, there was no significant change in protein levels of P21 and P16 in LPS group, but P21 and P16 increased significantly in siRNA-A20 group and siRNA-A20+LPS group (P < 0.05). β-galactosidase staining test showed that the number of positive cells increased significantly in siRNA-A20 treated groups (P < 0.05). The proportion of apoptosis in LPS group and siRNA-A20 group was higher than that of control group (P < 0.05), but lower than that of siRNA-A20+LPS group (P < 0.05). The result of TUNEL staining was consistent with that of AnnexinV-PI double labeling(P < 0.05). Apoptosis related protein BID and BAX increased (P < 0.05), and BCL2 decreased in siRNA-A20+LPS group (P < 0.05), while P-P65 increased significantly in siRNA-A20+LPS group(P < 0.05). Compared with siRNA-A20+LPS group, through the pretreatment of CAPE, protein levels of P-P65, P21, P16, BID and BAX decreased (P < 0.05), and the protein levels of BCL2 increased (P < 0.05). Conclusion Through down-regulation of zinc finger protein A20, LPS induces senescence and apoptosis of human degenerative nucleus pulposus cells through NF-κB signal pathway.

Keywords

• zinc finger protein a20
• human degenerative nucleus pulposus cells
• senescence
• apoptosis